Human Cytomegalovirus (hCMV) is a widespread, highly species-specific herpesvirus, causing significant morbidity and mortality in immunosuppressed or immunologically immature individuals.
Several recent reviews analyze hCMV biology and clinical manifestations (Landolfo S et al., 2003; Gandhi M and Khanna R, 2004; Soderberg-Naucler C, 2006a). This viral pathogen infects the majority of the population worldwide and is acquired in childhood, following the contact with a bodily fluid, since the virus enters through endothelial cells and epithelial cells of the upper alimentary or respiratory systems, or through the genitourinary system. Seropositivity to hCMV is more prevalent in underdeveloped countries or in geographical areas with lower income.
Following a primary infection, hCMV can persist in specific host cells of the myeloid lineage in a latent state, replicating and disseminating in many different cell types (haematopoietic cells, epithelial cells, endothelial cells, or fibroblasts) and escaping the host immune system. The hCMV infection is generally asymptomatic in healthy people since hCMV infection and dissemination is maintained under control by the immune system, but total hCMV clearance is rarely achieved. In fact, hCMV virus has developed efficient mechanisms that allow viral genome to remain in selected sites in a latent state.
Any situation that weakens immune functions, such as stress conditions or specific medical treatments, can lead to hCMV reactivation. Clinical manifestations of hCMV (such as retinitis, enterocolitis, pneumonitis, gastritis, or hepatitis) can occur following viral primary infection, reinfection, or reactivation. About 10% of infants are infected by the age of 6 months following transmission from their mothers via the placenta, during delivery, or by breastfeeding.
The hCMV virion consists of an icosahedral nucleocapsid which contains a linear, 230 kb-long, double-stranded DNA genome. The expression of hCMV genome is controlled by a complex cascade of transcriptional events that leads to the synthesis of more than 200 proteins involved in a large variety of biological activities involved in viral infection, latency, and replication (Britt W and Mach M, 1996).
The structural proteins form the virion envelope that is extremely complex and still incompletely defined. It includes glycoproteins that are homologues of structural proteins identified in other herpesviridae and that can form disulfide-linked protein complexes within the virion : gCI (including only gB), gCII (including gM and gN) and gCIII (including gH, gL and gO). The gB, gH, and gN genes have been also used for genotyping hCMV strains (Coaquette A et al.,2004; Dar L, 2007).
The glycoproteins gN and gM are the most abundant and, together with gH and gB, have been shown to be essential for the initial interaction between the hCMV envelope and host cell surface, and consequently for the production of infectious hCMV. For this reason, compounds targeting gB, gH, gN, and/or gM can efficiently inhibit hCMV infection by blocking the entry of circulating hCMV virions into the cells, following hCMV infection, reinfection, or reactivation.
Treatment of hCMV infections is difficult because there are few therapeutic options available. The presently available drugs compounds that inhibit viral replication (Ganciclovir, Cidofivir, Foscarnet, Maribavir and others drugs under development) produce a significant clinical improvement, but may suffer from poor oral bioavailability, low potency, the emergence of hCMV resistance (due to mutations in the viral targets), and dose-limiting toxicities (De Clercq E, 2003; Baldanti F and Gerna G, 2003; Gilbert C and Boivin G, 2005).
Novel means for preventing and treating hCMV infection are needed, especially for immunocompromised individuals, in transplantation settings, and in prenatal prevention. In fact, hCMV is a clinically important opportunistic pathogen in HIV patients and in organ transplant recipients, where it contributes to graft loss independently from graft rejection, resulting in morbidity and mortality (Puius Y and Snydman D, 2007). The increasing number of bone marrow and solid organ-transplant recipients raises the likelihood of hCMV clinical manifestations, such as hCMV retinitis (Wiegand T and Young L, 2006). Moreover, hCMV is the major infectious cause of birth defects (such as hearing loss, delayed development, or mental retardation) which are due to a congenital or perinatal hCMV infection transmitted by an hCMV-infected mother (Griffiths P and Walter S, 2005).
Thus, it is important to provide drugs for universal preemptive, prophylactic hCMV-specific treatments, for example for the prevention of hCMV disease in transplant recipients (Hebart H and Einsele H, 2004; Kalil A et al., 2005; Snydman D, 2006), in patients developing hCMV-related neuropathologies (Griffiths P, 2004) or in at risk pregnancies (Schleiss M, 2003), to prevent the vertical transmission and life-threatening hCMV infection to foetuses and neonates.
Moreover, pharmaceutical compositions against hCMV may be useful for the treatment of other, more widespread diseases (such as cardiovascular and autoimmune diseases, or some types of cancer), where hCMV is a possible cofactor and/or can be reactivated during immuniosuppressive treatments. For example, hCMV is now a human pathogen of growing importance for disorders such as long-term complications in tumour invasiveness and immune evasion since hCMV infection may have oncomodulatory effects on cell apoptosis, differentiation, and migration. In autoimmune or vascular diseases, hCMV infection may alter immune and inflammatory reactions (Cinatl J et al., 2004; Soderberg-Naucler C, 2006b).
An alternative way to prevent hCMV infection is vaccination, at the scope of providing protection in an array of high-risk patient populations. However, the correlation between vaccination and the resulting immune response is not fully understood and optimal hCMV vaccine strategy (using specific candidate antigens or live attenuated vaccines) seems depending on the patient population being targeted for protection. Therefore, prophylactic vaccination strategies are still under evaluation (McLean G et al., 2006; Schleiss M, 2005).
In view of the present limitations of pharmacological strategies for hCMV infections, the increasing knowledge of the host-hCMV relationship, and in particular on the hCMV-specific immune response, makes immune-based therapies good alternatives to substitute, or complement, existing therapies for the successful treatment of hCMV-associated complications (Gandhi M and Khanna R, 2004). Recently, a long-term protection from the lethal course of CMV infection in immunodeficient mice was achieved by transferring virus-specific memory B cells, suggesting that such cells may have a therapeutic utility (Klenovsek K et al., 2007).
An easier alternative to cell-based therapies can be passive immunotherapy, consisting in the administration to individuals of pharmaceutical compositions comprising therapeutic antibodies with a defined neutralizing activity against a human or viral antigen (e.g. hCMV).
This therapeutic approach has been designed on the antigen-binding and biological features of antibodies and antibody fragments directed against human or viral therapeutic targets (Dunman P and Nesin M, 2003; Keller M and Stiehm E, 2000). Passive immunotherapy has been introduced into clinical practice, rapidly expanding the opportunities for the treatment of a wide variety of diseases (including infectious diseases, immune-mediated diseases, and cancer). This approach can be particularly effective in patients whose immune system is unable to produce antibodies in the amounts and/or with the specificity required to block and/or eliminate the targeted molecule (Chatenoud L, 2005; Laffly E and Sodoyer R, 2005).
In the field of hCMV therapy, this approach is performed by administering intravenously human immunoglobulin preparations that are obtained by pooling human plasma with high titers of anti-hCMV antibodies, and commercialized for clinical uses (under the name of Cytotect or CytoGam). However, these products are only a partially satisfactory solution for blocking hCMV infection. In fact, this treatment is used in immunocompromised patients, mostly for pre-emptive treatment and prophylaxis where antivirals are often co-administered (Marasco W and Sui J, 2007; Nigro G et al., 2005; Bonaros N et al., 2004; Kocher A et al., 2003; Kruger R et al., 2003). Moreover, safety issues and shortage of such preparations are a growing concern, as reported in literature (Bayry J et al., 2007; Hamrock D, 2006).
Human recombinant antibodies that have high affinity for antigens expressed on hCMV envelope and are able to neutralize the infection would represent more appropriate drugs for passive immunization. In fact, several of the hCMV glycoproteins elicit strong host immune responses, including the production of virus-neutralizing antibodies, even though the stoichiometry of the envelope proteins is variable and may be altered to escape host immune response. This response is considered to be a key component of host immunity and represents a goal of both antibody and vaccine development.
Human monoclonal antibodies are the most preferable antibodies for clinical applications, due to the intrinsic limitations of murine monoclonal antibodies. However, the development of previously identified human antibodies for hCMV treatment (Matsumoto Y et al., 1986) has been interrupted since no clinical benefits were observed in studies that evaluated the efficacy of such antibodies, for example, in haematopoietic stem cell transplantation (Boeckh M et al., 2001), or in retinitis (Gilpin A et al., 2003). These failures trials warrant further studies aimed at selecting human monoclonal antibodies that more efficiently neutralize the widest variety of hCMV strains. The treatment of CMV infections would benefit from having more potent pharmaceutical compositions comprising human monoclonal antibodies that are purified from human B cells maintained in culture or produced as recombinant proteins that are expressed by human sequences introduced in mammalian cell lines.